Part:BBa_K2033000
N-dodecanoyl-L-homoserine lactone (C(12)-HSL) Sender- AubI
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 535
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 510
- 1000COMPATIBLE WITH RFC[1000]
Short Description
This is a synthase enzyme that produces N-dodecanoyl-L-homoserine lactone (C(12)-HSL).
Aub System Introduction
AHL quorum sensing functions within two modules. The first module, the "Sender," must be induced by certain environmental conditions, usually population density of surrounding organisms. This will begin production of the AHL by an AHL synthase, which is then detected by the second module, the "Receiver." The Receiver will cause the expression or silencing of certain genes to achieve the desired purpose of the communication, whether it is the production of GFP or to increase growth rate.
The Aub system was discovered as a result of a metagenomic soil study, in which the system was discovered. However, because the study was part of a metagenomic soil study, the specific bacterial origin is unknown. The Aub system produces a unique AHL molecule, which is shown below:
This AHL possesses an alkane tail, which is the primary recognition factor for AubR. The further characterization of this part is shown in the Design portion of this part.
Aub System
The original AubI part was sampled from soil and is thought to be from an unknown soil-borne bacteria. The bacteria was transformed into BL21 Competent E. coli cells to produce sufficient stock for future experiments. Of the 70 colonies that were produced, three samples were taken for verification. After mini-prepping, the gel verification affirmed the identity of the sample, as shown below:
After gel verification and sequencing, the AubI part was retransformed in BL21(DE3) E. coli and run in a 96-well plate from 580-610nm to measure mCherry production. This produced the curve below, indicating that the AHL is being produced by the sender, since mCherry production increased over an 8hour read time. This is because both the AHL and mCherry genes are found on the Sender.
Mass spectrometry was also used to characterize AubI. After purification of the AHL serum by HPLC, a PBwas combined with the sample and run using mass spectrometry. The comparison below between the negative control and the sample shows that a peak around 283.9 m/z appeared in the sample, which matches the predicted mass to charge ratio of the AubI AHL.
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